Apparatus and methods for analyzing samples

ABSTRACT

The present invention relates to apparatus, systems, and methods for analyzing biological samples. The apparatus, systems, and methods can involve using a vacuum source to pull microfluidic volumes through analytical equipment, such as flow cells and the like. Additionally, the invention involves using optical equipment in conjunction with the analytical equipment to analyze samples and control the operation thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.10/990,242, filed on Nov. 16, 2004, which claims priority to U.S.provisional patent application Ser. No. 60/589,170, filed on Jul. 19,2004, the disclosures of which are incorporated herein by reference intheir entirety. This application also incorporates herein by reference aU.S. patent application Ser. No. 10/990,167, filed Nov. 16, 2004.

TECHNICAL FIELD

The invention relates generally to apparatus, methods, and systems forhandling and analyzing microfluidic volumes and related biologicalmaterials. Additionally, the invention relates to optical equipment,such as lighting systems, for analyzing biological samples.

BACKGROUND

Generally, systems for analyzing a sample in a flow cell are pressuredriven fluidic systems using pressure pumps. Pressure driven fluidicssystems have several disadvantages. One disadvantage is that pressuredriven systems require the sample vessel to be sealably engaged to theflow cell assembly. This makes removal of the flow cell morecomplicated, because removal of the flow cell can produce hazardousaerosols. Pressure systems are also known to develop system leaks due tothe pressure and may require frequent replacement of lines and valves.Additionally, pressure driven systems can introduce contaminants intothe sample. Another disadvantage of pushing fluid through the system isthat air can become trapped in the system or air bubbles can beintroduced into the sample. Introduction of air into the pump can causecavitation resulting in shock to the system. Moreover, in pressuredriven systems, it is difficult to adequately purge the lines after eachsample has been tested. This can result in residual material being leftin the system when the next test is performed. Also, purging the systemusing air pressure tends to cause bubbling or foaming in the samples,which may introduce inaccuracies to the analysis.

The prior art vacuum driven systems that have been used to analyzesamples in a flow cell also have disadvantages. In these prior artsystems, a vacuum pump is directly connected to the flow cell. Again,the use of a pump can cause air bubbles to be introduced into the sampleand air trapped in the pump transmit shock to the system. Additionally,the continuous on and off cycle of the pump can result in uneven passageof a sample through the flow cell. Prior art vacuum systems are alsogenerally suited for passing multi-cell samples through the flow cell.Having a pump directly connected to the flow cell can negatively impactsingle-cell samples, in part, because of the shock transmitted to thesystem.

In analyzing microfluidic volumes and related biological materials usinga light source, it is desirable for the light source to hit the samplein such a way that results in total internal reflection fluorescence(“TIRF”). TIRF is an optical phenomenon that occurs when lightpropagating in a dense medium, such as glass, meets an interface with aless dense medium such as water. If the light meets the surface at asmall angle, some of the light passes through the interface (isrefracted) and some is reflected back into the dense medium. At acertain angle, known as the critical angle, all of the light isrefracted. However, some of the energy of the beam still propogates ashort distance into the less dense medium, generating an evanescentwave. The evanescent wave only penetrates about 100 nm into the medium.If this energy is not absorbed, it passes back into the dense medium.However, if a flourophore molecule is within the evanescent wave, it canabsorb photons and be excited. The excited fluorophores can be observedusing, for example, an intensified CCD camera. Accurately maintainingthe critical angle to obtain TIRF in a dynamic system is difficult.

SUMMARY OF THE INVENTION

The present invention involves using a vacuum source to pullmicrofluidic volumes through analytical equipment, such as flow cellsand the like. Generally, the invention includes a passive vacuum sourceand one or more valves and sensors for operating and monitoring theapparatus and methods. Additionally, the invention involves usingoptical equipment in conjunction with the analytical equipment toanalyze samples and control the operation thereof.

In one aspect, the invention relates to a lighting system including afirst light source for analyzing a sample of interest and a second lightsource. The first light source defines a first optical path thatintersects a sample of interest and the second light source operateswith the first light source for determining a position of the firstoptical path.

In various embodiments of the foregoing aspect, the first light sourceand the second light source operate simultaneously. The second lightsource may define a second optical path at least partially coaxial withthe first optical path. In one embodiment, the second light source isdirected to a position sensor for sensing an angle of reflection of thefirst optical path relative to the sample of interest. The position ofthe first optical path can be adjusted to vary the angle of reflectionin response to a signal from the position sensor. The position of thefirst optical path can be adjusted to obtain substantially totalinternal reflection of the first light source relative to the sample ofinterest.

Additionally, the first light source can have a wavelength from about390 nm to about 780 nm. In one embodiment, the second light source isinfrared light. The first light source and/or the second light sourcecan be a laser, a light emitting diode, or a lamp. In one embodiment,the system includes an imaging device for imaging the sample ofinterest. Further, the system can include a third light source foranalyzing the sample of interest. The third light source can define athird optical path at least partially coaxial with the first opticalpath. The first light source and the third light source can be operatedsimultaneously. The second light source may be used to continuouslymonitor the position of the first optical path. In one application, thelight system can be adapted for use in a single molecule sequencingsystem.

In another aspect, the invention relates to a method of substantiallymaintaining total internal reflection for a sample of interest. Themethod includes the steps of providing a first beam of light forintersecting with the sample of interest, providing a second beam oflight for determining a position of the first beam of light, directingthe second beam of light onto a position sensor, and adjusting theposition of the first beam of light in response to a signal from theposition sensor to vary an angle of reflection of the first beam oflight with respect to the sample of interest to substantially maintaintotal internal reflection.

In various embodiments, the first beam of light is at least partiallycoaxial with the second beam of light. The first beam of light is foranalyzing the sample of interest. In one embodiment, the first lightsource has a wavelength from about 390 nm to about 780 nm. The secondlight source may be infrared light. The method may also include thesteps of continuously monitoring the position of the first beam of lightand adjusting the angle of reflection in response thereto tosubstantially maintain total internal reflection.

In another aspect, the invention relates to a system for analyzing asample. The system includes a flow cell, a passive vacuum source forpulling a volume through the flow cell, a lighting system forilluminating the sample in the flow dell, and an optical instrument forviewing the sample in the flow cell. The lighting system can be of thetype described hereinabove. In one embodiment, the volume includes thesample or agents for reacting with the sample, which may be predisposedon or within the flow cell. Alternatively or additionally, the samplemay adhere to or come to rest within the flow cell while the volumepasses therethrough. In one embodiment, the volume and/or sample ismoved through the flow cell by gravity. For example, the head pressureon the volume within an inlet to the flow cell is sufficient to move thevolume through the flow cell.

In various embodiments of the foregoing aspect, the system includes astage for receiving the flow cell, where the stage is movable in atleast one direction. In one embodiment, the stage is movable in twoorthogonal directions. The system may also include an image capturedevice for capturing an image of the sample. The image capture devicecan be a charge coupled device (CCD), a complementary metal oxidesemiconductor device (CMOS), a charge injection device (CID), or a videocamera. Additionally, the system could include a processor forcollecting and processing data generated by the system, storage forstoring the data, and means for displaying at least one of the data andthe sample.

In another aspect, the invention relates to an apparatus for handlingmicrofluidic volumes, such as biological samples for analysis. Theapparatus can include the aforementioned passive vacuum source and flowcell. The microfluidic volume is pulled through the flow cell by thepassive vacuum source. In one embodiment, the passive vacuum sourceincludes a pump, a pump driver, such as an electric motor, and areservoir. The pump can be connected to the reservoir and then operatedto evacuate the reservoir, thereby creating a vacuum within thereservoir. In one embodiment, the vacuum pressure is from about 1″ Hg toabout 29″ Hg. The vacuum pressure can be adjusted to vary the speed atwhich the microfluidic volume passes through the flow cell.

In various embodiments of the foregoing aspects, the apparatus/systemcan be used for single molecule detection. In one embodiment, the flowcell includes a surface for receiving a nucleotide. For example, theflow cell can include a bound nucleotide and a primer bound to thenucleotide and/or the flow cell. In particular, the flow cell caninclude a slide and a coverslip, where the nucleotide and/or the primerare bound to at least one of the slide and the coverslip. Additionally,the flow cell can include a channel for pulling the microfluidic volumetherethrough.

In some embodiments of the foregoing aspects, the ratio of a volume ofthe reservoir and the microfluidic volume is between about 1,000:1 andabout 2,000,000:1, or between about 50,000:1 and about 1,000,000:1, orabout 200,000:1. Further, the apparatus can include valving disposedbetween the various components thereof. For example, the apparatus caninclude a valve disposed between the vacuum source, for example thereservoir, and the flow cell, wherein the valve includes an openposition to connect the flow cell to the vacuum source and a closedposition to isolate the flow cell from the vacuum source. The apparatuscan also include a vacuum pressure indicator connected to the reservoir.Moreover, the apparatus can further include optical equipment foranalyzing material within the flow cell after exposure to themicrofluidic volume.

In another aspect, the invention relates to a method of detecting singlemolecules. The method includes the steps of depositing a samplecomprising single molecules into a flow cell, the flow cell treated toidentify specific molecules; applying a vacuum to the flow cell; pullingthe sample through a channel defined by the flow cell; and viewing theflow cell after exposure to the sample to identify the molecules exposedto the flow cell.

In another aspect, the invention relates to a method of detecting singlemolecules. The method includes the steps of providing a flow cell thatdefines a channel that is treated to identify specific molecules,applying a vacuum to the channel to pull a sample through the channel,the sample comprising single molecules, and viewing the sample in thechannel to identify the single molecules.

Various embodiments of the foregoing methods include the step ofremoving the vacuum from the flow cell after pulling the sample throughthe channel. The step of applying a vacuum can include exposing the flowcell to a passive vacuum source. In various embodiments, the sampleincludes a microfluidic volume including nucleotides. Additionally, theflow cell can include at least one of a slide and a coverslip treated tobind with a specific nucleotide. Further, the step of viewing the flowcell can include illuminating the flow cell with a lighting system, suchas that described hereinabove. The step of viewing the flow cell canalso include using an image capture device. In one embodiment, aprocessor is used to control the operation of the method. The processorcan be used for collecting and processing data generated during themethod. The method can further include the step of displaying at leastone of the flow cell and the data.

In another embodiment, single nucleotide detection is accomplished byattaching template nucleic acids to a flow cell in the presence of aprimer for template-dependent nucleic acid synthesis. Using a deviceaccording to the invention, a vacuum is created across the flow cell forintroduction of reagents for template-dependent nucleic acid synthesis.For example, once template/primer pairs are bound to the surface of theflow cell, reagents comprising labeled or unlabeled nucleotides and apolymerase to catalyze nucleotide addition are added via an entry port.The vacuum is switched on and the reagents are exposed to the flow celland then exit via an exit port to the reservoir. After a wash step,complementary nucleotides added to primer are detected. Preferably,reagent nucleotides are labeled with, for example, a fluorescent dye.Such dyes are observed using sight microscopy. For example, cyanine dyes(cyanine-3 or cyanine-5) are useful for optical detection ofincorporated nucleotides. Using optically-detectable labels, nucleicacid sequencing is conducted on a single molecule level. This means thatindividual template nucleic acids are positioned on the flow cell suchthat each is individually optically resolvable. The location of thetemplates is determined by, for example, the use of dye-labeled primersthat hybridize to individual templates. Labeled nucleotides are flowedacross the flow channel using the mechanisms described herein underconditions that allow complementary nucleotide addition to the primer.Once incorporated, the label is detected by excitation of the dye at theappropriate wavelength and by using an emission filter for detection ofthe emission spectrum. Emissions that occur at a location known tocontain a template indicate incorporation of the labeled base at thatposition. By conducting these steps multiple times, a sequence iscompleted. Single molecule sequencing techniques are described inBraslaysky, et al., PNAS (USA), 100: 3960-3964 (2003) and copending U.S.patent application Ser. No. 09/707,737, each of which is incorporated byreference herein.

In another aspect, the invention relates to a flow cell for analyzingsingle molecules, such as nucleotides. The flow cell includes a slide, acoverslip, and a gasket disposed between the slide and the coverslip.The slide, the coverslip, and the gasket define a microfluidic channelfor passing single molecules under vacuum. In various embodiments, theflow cell includes a nucleotide hound to the slide and/or the coverslip.In addition, the flow cell can include a primer bound to at least one ofthe nucleotide, the slide, and the coverslip. In one embodiment, theslide includes a plurality of nucleotides bound thereto.

In another aspect, the invention relates to a slide for use with a flowcell. The slide can include at least one nucleotide bound to a surfaceof the slide. The slide can be disposed within the flow cell. The slidecan further include a primer bound to at least one of the slide and thenucleotide. In addition, the slide can include a plurality ofnucleotides bound thereto.

In another aspect, the invention relates to a coverslip for use with aflow cell. The coverslip includes at least one nucleotide bound to asurface of the coverslip. The coverslip can be disposed within the flowcell. The coverslip can further comprise a primer bound to at least oneof the coverslip and the nucleotide. In one embodiment, the coverslipincludes a plurality of nucleotides bound thereto.

These and other objects, along with advantages and features of thepresent invention herein disclosed, will become apparent throughreference to the following description, the accompanying drawings, andthe claims. Furthermore, it is to be understood that the features of thevarious embodiments described herein are not mutually exclusive and canexist in various combinations and permutations.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference characters generally refer to the sameparts throughout the different views. Also, the drawings are notnecessarily to scale, emphasis instead generally being placed uponillustrating the principles of the invention. In the followingdescription, various embodiments of the present invention are describedwith reference to the following drawings, in which:

FIG. 1 is a schematic representation of one embodiment of an apparatusfor handling microfluidic volumes in accordance with the invention;

FIG. 2 is a schematic representation of an alternative embodiment of anapparatus for handling microfluidic volumes in accordance with theinvention;

FIG. 3 is a schematic representation of another alternative embodimentof an apparatus for handling microfluidic volumes in accordance with theinvention;

FIG. 4A is a pictorial representation of one possible configuration ofthe apparatus of FIG. 1;

FIG. 4B is a pictorial representation of a portion of the apparatus ofFIG. 4A;

FIG. 5 is a plan view of a portion of the apparatus of FIG. 4A;

FIG. 6 is a side view of a portion of the apparatus of FIG. 4A;

FIG. 7A is a block diagram of a system in accordance with one embodimentof the invention;

FIG. 7B is a pictorial representation of the system of FIG. 7A;

FIG. 8 is a flow chart depicting one mode of operation of a method ofhandling microfluidic volumes in accordance with the invention;

FIG. 9 is a plan view of a flow cell in accordance with one embodimentof the invention;

FIG. 10 is a cross-sectional view of the flow cell of FIG. 9 taken atline 10-10;

FIG. 11 is an exploded view of the flow cell of FIG. 9; and

FIGS. 12A and 12B are schematic representations of a lighting system inaccordance with one embodiment of the invention.

DESCRIPTION

Embodiments of the present invention are described below. It is,however, expressly noted that the present invention is not limited tothese embodiments, but rather the intention is that modifications thatare apparent to the person skilled in the art are also included. Forexample, many of the following embodiments are described with referenceto pulling microfluidic volumes through a flow cell, however, thepresent invention can also be applied to pulling fluids through othertypes of analytical equipment, such as, for example, flow cytometers andchemical analyzers. Further, the apparatus can be used as part of asystem for detecting single molecules by, for example, optical detectionof single nucleotides.

In one embodiment, the apparatus 10 includes a vacuum source 12, anisolation valve 20, and a flow cell 30. In the embodiment depicted inFIG. 1, the vacuum source 12 is passive and includes a vacuum pump 14, adrive motor 16, and a reservoir 18. Alternatively, the vacuum source 12could be non-passive, where the vacuum pump 14 is directly connected tothe flow cell (see, for example, FIG. 3). In one embodiment, the vacuumpump 14 is a compact rotary vane type pump; however, the pump size andtype will be selected to suit the particular application. For example,the pump could be a piston, gear, or diaphragm type pump. Further, thepump size will depend on the operating parameters of the apparatus 10,for example, the larger the pump capacity, the quicker the pump 14 willevacuate the reservoir 18. The drive motor 16 in one embodiment is a 12volt DC electric motor; however, the motor size and type will beselected to suit the particular application. For example, larger flowsmay require a larger pump, which in turn may require a larger motor.Further, the pump 14 can be uni- or bi-directional and can be coupled tothe motor 14 directly or via a flexible coupling or other means known toone of skill in the art. In a particular embodiment, the pump 14 andmotor 16 are supplied as an assembly, such as model no. 50200 availablefrom Thomas Pumps and Compressors of Shebogan, Wis.

The reservoir 18 in one embodiment is a four liter bottle, such asNalgene® model no. 2125-4000 available from Nalge Nunc International ofRochester, N.Y. The reservoir size will be selected to suit a particularapplication and, as will be discussed in greater detail below, istypically substantially larger than the microfluidic volume to be pulledby the vacuum source 12. In addition, the reservoir material can be ametal, a polymer, glass, or combinations thereof. In particular, thereservoir material should be compatible with the microfluidic volume 32.Also, the reservoir 18 should be capable of withstanding the pressuresto which the reservoir 18 is exposed. For example, the reservoir 18should be able hold a vacuum with minimal leakage and withoutcollapsing.

The apparatus 10 shown in FIG. 1 includes three valves, 20A, 20B, 20C(collectively 20). The valves 20 shown are two position, threeconnection type solenoid valves, such as model no. LHDA1233115Havailable from the Lee Co. of Westbrook, Conn. The solenoids, whichactuate the valves, are energized by 12 volt DC; however, other voltagescan be used and the valves can be actuated hydraulically, pneumatically,or manually. Additionally, the valve type and configuration can beselected to suit a particular application. For example, the valves canbe two position, two connection or two position, four connection.

The first valve 20A is located between the reservoir 18 and the pump 14.In the unactuated state, the valve 20A isolates the reservoir 18 fromthe pump 14. The pump inlet 40 is connected to the atmosphere, while thereservoir outlet 42 is closed. Alternatively, the pump inlet 40 could beclosed. When the first valve 20A is actuated, for example by energizingthe solenoid, the valve 20A changes position, thereby connecting thepump inlet 40 to the reservoir outlet 42 and allowing the pump 14 (whenrunning) to pull a vacuum on the reservoir 18. In one embodiment, thevacuum pressure is between about 1″ Hg and about 29″ Hg, preferablybetween about 2″ Hg and 15″ Hg, and more preferably between about 5″ Hgand about 6″ Hg; however, the vacuum pressure can be varied to suit aparticular application. Generally, the greater the vacuum pressure, thefaster the microfluidic volume 32 will be pulled through the flow cell.In some cases, a fast flow is desirable to reduce the amount of residueleft within the flow cell 30 from the microfluidic volume 32.

The second valve 20B is located between the reservoir 18 and the flowcell 30. In the unactuated state, the valve 20B isolates the reservoir18 from the flow cell 30. The reservoir inlet 44 is closed, while theflow cell outlet 46 is connected to the atmosphere. Alternatively, theflow cell outlet 46 could also be closed. When the second valve 20B isactuated, the valve 200 changes position, thereby connecting the flowcell outlet 44 to the reservoir inlet 46, which results in the vacuumwithin the reservoir 18 pulling the volume of material 32 through theflow cell 30. The vacuum pressure within the reservoir 18 determines thespeed at which the volume 32 is pulled through the flow cell 30.

Optionally, a third valve 20C, as shown in FIG. 1, is connected to thereservoir 18 and is used to vent the reservoir 18. The optional thirdvalve 20C could be located at a different location on the apparatus 10to perform a different function. Alternatively or additionally, multiplevalves 20 can be used in conjunction with multiple flow cells 30. Forexample, the apparatus 10 can include ten flow cells 30, or otheranalytical equipment, each connected in series with a valve 20 and thereservoir 18 (see, for example, FIG. 2).

The flow cell 30 is coupled to the vacuum source 12, as described above.Multiple flow cells 30, or other analytical equipment, can be connectedto the vacuum source 12 either in series or in parallel (see, forexample, FIG. 2). In one embodiment, the flow cell 30 is a Focht ChamberSystem (model no. FCS2) available from Bioptechs of Butler, Pa.Alternatively, a customized flow cell system may be used. The flow cell430 depicted in FIGS. 9-11 is a customized flow cell and will bedescribed in greater detail with respect to FIGS. 9-11

Further depicted in FIG. 1 is a pipette 50 for introducing themicrofluidic volume 32 to the apparatus 10; however, other types ofvessels can be used for introducing the volume 32 to the apparatus 10.For example, a cuvette or beaker could be used. The pipette 50 ispositioned directly over the flow cell inlet 48. In one embodiment, themicrofluidic volume 32 includes single molecules for use in sequencingdeoxyribonucleic acid (DNA). In one embodiment, the pipette 50 canmanually or automatically dispense individual microfluidic volumes inthe range of about 2 microliters (μl) to about 2 milliliters (ml),preferably about 10 μl to about 100 μl, and more preferably about 20 μl.Further, the pipette 50 can be handled robotically to, for example,position the pipette 50 relative to the flow cell inlet 48, receive andmix materials within the pipette 50, and/or dispense precisely themicrofluidic volume 32 based on time and/or volume.

The apparatus 10 further includes a pressure indicator 60, such as modelno. DPG1000B-30INHGVAC available from Omega Engineering, Inc. ofStamford, Conn. The indicator 60 is used to measure the vacuum pressurewithin the reservoir 18; however, additional indicators can be used tomeasure the pressure at other locations in the apparatus 10, forexample, the flow cell outlet 46. The indicator 60 can be a pressuregauge, a pressure transducer, and/or pressure switch, with or without areadout. For example, the pressure transducer could include a digitalreadout of the actual vacuum pressure within the reservoir 18 and/or thepressure switch can activate an alarm if the pressure within thereservoir 18 reaches a threshold value.

The apparatus 10 depicted in FIG. 1 also includes an optional controller70. The controller 70 includes the electronic controls for operating,for example, the vacuum source 12 and valves 20 by, for example, acomputer 68 and related software. The apparatus 10 can send and receivedata directly or via the controller 70 to the computer 68. The computer68 can be a conventional computer system including a processor, harddrive, RAM, a video monitor, and a keyboard, as may be found in alaboratory setting. The computer 68 can interact with the controller 70to store and process data as necessary to operate the apparatus 10.Alternatively or additionally, the controller 70 can include an internaldata processor. Alternatively, the apparatus 10 can be controlledmanually. The controller 70 shown is a switch and sense type controlleravailable from Measurement Computing Corporation of Middleboro, Mass.The exact controller configuration will be selected based on, forexample, the number of inputs and outputs required and the type ofequipment to be controlled. In one embodiment, the controller 70 caninclude the logic for cycling the pump 14 and motor 16 on and off andactuating the valves 20 based on predetermined time intervals and/or inresponse to signals from sensors. The controller can also supply thenecessary power to the various components of the apparatus 10.

FIG. 2 depicts schematically an alternative embodiment of an apparatus110 in accordance with the invention. The apparatus 110 is similar tothe apparatus 10 described hereinabove with respect to FIG. 1; however,the apparatus 110 shown in FIG. 2 includes multiple flow cells 130,130′, 130″ and corresponding second valves 120B, 120B′, 120B″ arrangedin a parallel configuration. As described above, the apparatus 110includes a passive vacuum system 112 including a pump 114, a motor 116,and a reservoir 118; a first valve 120A; a pressure indicator 160; and acontroller 170.

The multiple flow cells 130, 130′, 130″ and the corresponding secondvalves 120B, 120B′, 120B″ are arranged in parallel to facilitate runningmultiple operations either simultaneously or sequentially. For example,the user can run three different operations without having to changeset-ups between operations. The large ΔV between the reservoir 118 andthe microfluidic volumes 32, 32′, 32″ facilitates multiple operationswithout any degradation in performance. Alternatively or additionally,the flow cells 130 could be arranged serially; however, seriallyarranged flow cells 130 would have to be operated simultaneously and mayimpact the adjacent flow cell(s) 130.

FIG. 3 depicts schematically another alternative embodiment of anapparatus 210 in accordance with the invention. The apparatus 210 issimilar to the apparatus 10, 110 described hereinabove with respect toFIGS. 1 and 2; however, the apparatus 210 shown in FIG. 3 does notinclude a reservoir. The apparatus 210 includes a non-passive vacuumsystem 212 including a pump 214 and a motor 216, where the pump 214 isdirectly connected to the flow cell 230 via a single valve 220. Theapparatus 210 further includes a pressure indicator 260 located betweenthe pump inlet 240 and the flow cell outlet 246, and a controller 270.

FIG. 4A is a pictorial representation of one possible configuration ofthe apparatus 10 depicted schematically in FIG. 1. The vacuum system 12,valves 20, and indicator 60 are mounted on a breadboard 72; thereservoir 18 is free-standing adjacent to the breadboard 72; and theflow cell 30 is disposed on a microscope type stage 52 adjacent to thebreadboard 72. The breadboard 72 is mounted on top of the controller 70via stand-offs 74 and screws 76 located at the four corners of thebreadboard 72. Also mounted on the breadboard 72 are push-buttons 56 foroperating the valves 20, and the electrical and fluidic connections forthe various components.

As shown in FIG. 4A, the apparatus 10 uses tubing 54 to connect thevarious components, for example, the pump 14 and reservoir 18. In oneembodiment, the tubing 54 is capillary type tubing, which can beobtained from, for example, Polymicro Technologies, LLC of Phoenix,Ariz. Alternatively or additionally, conventional polymer tubing can beused, for example, ⅛″ outside diameter nylon, such as Nylotube®available from New Age Industries, Inc. of Southampton, Pa. The size,type, and material of the tubing can be selected to suit a particularapplication. For example, metallic tubing may be undesirable forbiological materials and the size of the tubing 54 should be selectedbased on the flow parameters of the microfluidic volumes. For example,the inside diameter of the tubing 54 should be sufficient to preventturbulent flow of the microfluidic volume therethrough.

Moreover, the apparatus 10 can include various optical components, suchas a microscope objective, a camera, and multiple light sources foroptically analyzing the contents of the microfluidic volume 32 and/orthe operation of the apparatus 10. Additionally, the flow cell 30 can belocated on a microscope type stage 52 for optical viewing by the user.In one embodiment, the stage 52 can be moved in the X, Y, and/or Zdirections to position the flow cell 30 relative to the opticalcomponents. In an alternative embodiment, the flow cell 30 is securedwithin a stationary fixture. Alternatively or additionally, the opticalcomponents can be movable in the X, Y, and/or Z directions. Theapparatus 10 can also include additional sensors for monitoring variousoperations of the apparatus 10. For example, the apparatus 10 couldinclude an optical sensor for monitoring the level of the microfluidicvolume 32 within the flow cell inlet 48.

FIG. 4B is a pictorial representation of a portion of the apparatus 10shown in FIG. 4A. Specifically, FIG. 4B depicts an enlarged view of theflow cell 30 from the side opposite that shown in FIG. 4A. The flow cellinlet 48 is shown open and unobstructed. In operation, there would be apipette located above the flow cell inlet 48. The pipette would containand dispense the microfluidic volumes to be pulled through the flow cell30. Shown above the flow cell inlet 48 is a camera 80 that can be usedto display an image of the flow cell inlet 48 and the fluid flowtherethrough to the user on, for example, an optional video monitor.Alternatively or additionally, the image can be used in conjunction witha sensor to send a signal to the controller 70 to, for example, closethe second valve 20B. The flow cell outlet 46 is shown with a fittingand capillary tubing running therefrom. The fitting 74 is a conventionaltype of fitting that can be used to connect the tubing to the flow celloutlet 46, for example, a nut and ferrule type fitting. The tubing runsto the second valve 20B (see FIG. 4A). Shown adjacent to the flow cell30 is a heater 58 that can be used to heat the various components, forexample the flow cell 30, as needed to carry out a particular operation.

The apparatus 10 will be further described with reference to FIGS. 4A,4B, 5, and 6. The pump 14 and motor 16 are mounted to the breadboard 72by a bracket 74. The three valves 20 are also secured to the breadboard72. The pump 14 has two connections; the inlet 40 and an outlet 41. Theoutlet 41 is open to the atmosphere, but could include an exhaust filter24 (FIG. 1) or be plumbed to a remote location. The inlet 40 is plumbedto an outlet 43 on the first valve 20A via the tubing 54. The inlet 45of the first valve 20A is than plumbed to the reservoir 18. Theconnections between the pump 14, valves 20, and reservoir 18 are pushtype fittings, where the tubing 54 is pushed over the fittings andsecured by friction and/or barbs. Other types of fittings are alsocontemplated and considered within the scope of the invention.

An outlet 47 on the second valve 20B is plumbed to the reservoir 18. Aninlet 49 on the second valve 20B is plumbed to the flow cell 30. Thethird valve 20C is optional in the depicted configuration and is,therefore, not shown plumbed. The pressure indicator 60 includes aninlet 51 that is plumbed to the reservoir 18 to continuously monitor thevacuum pressure therein.

Each of the valves 20 and the motor 16 include electrical connections53. The electrical connections 53 are wired to the controller 70 forconnection to the necessary power source(s) and control logic. The pushbuttons 56A, 56B, 56C, 56D (collectively 56) also include electricalconnections that are wired to the valves 20, motor 16, and controller70. The controller 70 includes an electrical connection 78 forconnecting the controller 70 to the computer 68 (sec FIG. 1). Thecontroller 70 may include an additional connection for connecting to anexternal power source. In one embodiment, the electrical connection 78is a USA connection. Alternatively or additionally, the controller 70could include an IEEE 1394 connection, such as the FIREWIRE® brand soldby Apple Computer, Inc. The controller 70 can further include a powerswitch and indicators, either alone or as part of a user interface.

In the embodiment shown, the push buttons 56 are used to run the motor16, which drives the pump 14, and to actuate the valves 20 by energizingthe valve solenoids 21. Specifically, the first push button 56A, whenpushed, energizes the motor 16, thereby causing the pump 14 to pull avacuum. The second push button 56B, when pushed, energizes the firstvalve solenoid 21A, thereby connecting the pump 14 to the reservoir 18.When both push buttons 56A, 56B are pushed, the pump 14 evacuates theair out of the reservoir 18, thereby creating a vacuum within thereservoir 18. The third push button 56C, when pushed, energizes thesecond valve solenoid 21B, thereby connecting the reservoir 18 to theflow cell 30. The fourth push button, when pushed, energizes the thirdvalve solenoid 21C, thereby actuating the third valve 20C. The apparatus10 can include additional valves and push buttons as required by thespecific configuration. In addition, other types of switches could beused to operate the various components, as opposed to the push buttonsshown. For example, toggle type switches could be used.

FIG. 7A depicts schematically an embodiment of a system 300 inaccordance with the invention that includes an apparatus 310 andauxiliary components in accordance with the invention. FIG. 7B depictsone possible arrangement of the various components of the system. Theauxiliary components include a lighting/optics module 320, a microscopemodule 330, and a computer module 340. Generally, in one embodiment, thelighting/optics module 320 includes multiple light sources and filtersto provide light to the microscope for viewing and analysis. The lightis reflected onto, for example, a flow cell 312 seated on the microscopemodule 330 (see FIG. 7B). The light can be multiple wavelengths, forexample, one wavelength for viewing and another wavelength for analysis.A particular lighting/optics module 600 is described with respect toFIGS. 12A and 1213.

The microscope module 330 includes hardware for holding the flow cell312 and moving the microscope stage and an imaging device, such as acamera. In some embodiments, the microscope module 330 is a part of theapparatus 310. The computer module 340 includes the memory andprocessors necessary for operating the various modules and a userinterface for operating the system 300. The modules communicate with oneanother as shown by the arrows in FIG. 7A. For example, the computermodule 340 may send a signal to the lighting/optics module 320 based ona user input to, for example, send a red light to the microscope module330 to illuminate the flow cell. The computer module 340 can also sendand receive signals from the microscope module 330 to change and monitorthe position of the microscope stage or other operational parameters.Additionally, the computer module 340 can send and receive signals fromthe apparatus 310 to open and close valves.

As shown in FIG. 7B, the various components of the system 300 aremounted on a laboratory bench 302 in close proximity to one another;however, the arrangement of the various components can vary to suit aparticular application and/or environment. The microscope module 330includes a stage 332 for positioning the flow cell 312 or other item tobe analyzed, a camera 334, and optics 336. Generally, a microscope, suchas model no. TE2000 from Nikon Instruments, Inc. of Melville, N.Y., issuitable for use with the system 300; however, the type of microscopeused can be selected based on the particular application and the natureof the sample to be analyzed.

The computer module 340 includes a processor 342, a video monitor 346,and a user interface 344, such as a keyboard and mouse for interactingwith the system 300. In one embodiment, the camera 334 sends images tothe computer module 340 for analysis and/or display on the video monitor346. The lighting/optics module 320 of the system 300 includes anarrangement of light sources 342, 344 and filters 346 and mirrors 348for conditioning the light emitted by the light sources 342, 344. Thearrangement of the components will vary to suit a particular applicationand/or environment. The lighting/optics module 320 supplies conditionedlight to the microscope module 330 for the viewing and analysis of thesample disposed therein.

FIG. 8 represents the basic operation 500 of an apparatus in accordancewith one embodiment of the invention. Generally, a user monitors thevacuum condition within the reservoir (step S10). If, for example, thevacuum level is not within set limits, the user can increase the vacuumpressure within the reservoir by operating the vacuum pump (Steps S20,530). Once the vacuum pressure is within the set limits, the user candeposit a sample (e.g., a microfluidic volume) into the flow cell inlet(Step S40). Subsequently, the user will open the flow cell outlet to thereservoir, thereby pulling the sample through the flow cell (Step S50).Once the user or the controller determines that the flow cell inlet isempty (Step S60), the connection between the flow cell outlet and thereservoir is closed (Step S70). The user and or controller will maintainthe connection between the flow cell outlet and the reservoir open untilthe flow cell inlet is empty, as it is desirable to pull essentially allof the sample through the flow cell to prevent contaminating subsequentoperations. If there are additional samples to be pulled through theflow cell (Step S80), the basic operation is repeated until there are nomore samples, at which time the operation is ended (Step S90).Alternatively or additionally, the sample to be analyzed is containedwithin the flow cell, where the sample is exposed to the material orvolume of material pulled through the flow cell, thereby causing areaction or otherwise effecting the sample within the flow cell.

More specifically, in operation, the user creates a vacuum in thereservoir 18 by, for example, operating the pump 14 and motor 16 andactuating the first valve 20A isolating the pump 14 from the reservoir18. Once the desired vacuum is reached, for example about 6″ Hg, thefirst valve 20A is deactuated and the pump 14 and motor 16 are stopped.Next, the pipette 50 deposits a microfluidic volume 32 within the flowcell inlet 48 and, subsequently, the second valve 20B is actuated,thereby connecting the vacuum reservoir 18 to the flow cell outlet 46and pulling the microfluidic volume 32 through the flow cell 30 and intothe reservoir 18, thus resulting in a transient exposure of themicrofluidic volume and its contents to, for example, nucleotides thatare held within the flow cell. Furthermore, the sample or volume can bedriven through the flow cell by virtue of gravity, specifically the headof the volume held within the flow cell inlet or pipette. Once themicrofluidic volume 32 leaves the flow cell inlet 48, the second valve20B is closed, thereby removing the vacuum pressure from the flow cell30. Generally, the second valve 20B should be open only long enough topass the microfluidic volume 32 through the flow cell 30. If the valve2013 is open too long, air and bubbles can be pulled into the flow cell30; if not open long enough, a portion of the volume 32 will remain inthe flow cell 30, which could contaminate subsequent operations.Subsequently, the sample can be viewed and analyzed as desired.

In operation, it is desirable for the ratio of the reservoir volume 18to the microfluidic volume 32 to be very large. For example, the ratiocan be front about 1000:1 to about 2,000,000:1, preferably from about50,000:1 to about 1,000,000:1, and more preferably about 200,000:1. Inone embodiment, the reservoir 18 is about 4 liters (l) and themicrofluidic volume is about 20 μl, thereby resulting in a ratio ofabout 200,000:1. The exact ratio will depend on, for example, theleakage rate of the reservoir, the size of the microfluidic volume, andthe number of operations to be performed. A particularly large ratioresults in the operation of the apparatus 10 being substantiallyunaffected by leakage and/or the number of microfluidic volumes 32pulled through the flow cell 30, because the reservoir volume undervacuum is so great relative to the volumes being absorbed by thereservoir, the change in volume is negligible. For example:

P₁V₁=P₂V₂, where

P₁=the vacuum pressure within the reservoir prior to adding themicrofluidic volume (ΔV);

V₁=the volume within the reservoir prior to adding ΔV;

P₂=the vacuum pressure within the reservoir after adding ΔV; and,

V₂=the volume within the reservoir after adding ΔV.

Because V₁ is so large relative to ΔV, V₁ is substantially equal to V₂.Therefore, P₁ is substantially equal to P₂.

The valves 20, pipette 50, and pump 14 can be operated manually orautomatically. For example, the second valve 2013 can be programmed toactuate (i.e., open) for “x” seconds after the pipette 50 deposits thevolume 32 into the flow cell inlet 48 and deactuate (i.e., close) at theend of a set time period. In one embodiment, the time period can beadjusted to accommodate different volumes 32. In an alternativeembodiment, an optical sensor can be used to actuate and/or deactuatethe second valve 20B. For example, the second valve 2013 can be actuatedafter the optical sensor senses that the appropriate volume 32 has beendeposited into the flow cell inlet 48 and deactuated after the sensorsenses that the flow cell inlet 48 is empty. In one embodiment, thesensor(s) will send a signal to the controller 70, which in turn outputsthe appropriate response to the signal, e.g., deactuate the second valve2013. Additionally, the pressure sensor 60 can be used to control thefirst valve 20A and the pump 14. For example, if the pressure sensor 60senses that the vacuum in the reservoir 18 has degraded below athreshold value, the controller 70 can turn on the pump 14 and motor 16and actuate the first valve 20A to increase the vacuum in the reservoir18.

FIGS. 9, 10, and 11 depict the customized flow cell 430. The flow cell430 is similar to the Focht Chamber System and includes a connectionring 436, an upper gasket 438, a slide 440, a lower gasket 442, acoverslip 444, and a locking base 428. Also shown is an optional heater458. The connection ring 436 sits on top of the various components andwhen seated and locked in the base 428 seals the components in place. Itis desirable to operate the flow cell 430 by pulling a volume throughunder vacuum, as opposed to a pushing the volume through by positivepressure. Positively pressurizing the flow cell 430 may result in theslide 440 and/or coverslip 444 being bowed outwardly, contaminationbeing trapped between the gaskets 438, 442 and the slide 440 and/orcoverslip 444, or otherwise compromising the integrity of the flowcell's structure. By using vacuum, the contact areas between the gaskets438, 442 and the slide 440 and coverslip 444 are maintained, therebyeliminating the possibility of contamination collecting in those contactareas.

The connection ring 436 houses the flow cell inlet 448 and the flow celloutlet 446. In the embodiment shown, the inlet 448 and the outlet 446are machined through the ring 436. The inlet 448 is a conical shapedrecess and the outlet 446 is a threaded connection for accepting afitting. The conical shaped inlet 448 is as large as possible tofacilitate viewing the flow of any microfluidic volumes depositedtherein. The connection ring 436 also defines a viewing area 432 wherethe slide 440 and coverslip 444 are visible. Further, the connectionring 436 should be made of a material that is dimensional stable,compatible with the microfluidic volumes passed therethrough, and towhich any substances within the microfluidic volumes will not stick.Such materials include, for example, polyetheretherketone, sold by PLCCorporation under the trademark PEEK®; polyoxymethylene, sold by DuPontunder the trademark Delrin®; polytetrafluoroethylene, sold by DuPontunder the trademark Teflon®; and ethlene-chlorotrifluorethylene, sold byAllied Chemical Corporation under the trademark Halar®.

The upper gasket 438 provides the seal between the slide 440 and theconnection ring 436. In the embodiment shown, the upper gasket 438 has athin annular shape; however, the size and shape of the upper gasket 438will vary to suit a particular application. The lower gasket 442provides the seal between the slide 440 and the coverslip 444. In theembodiment shown, the lower gasket 442 covers a substantial portion ofan upper surface of the coverslip 444. In particular, the lower gasket442, along with a lower surface 441 of the slide 440, and an uppersurface 445 of the coverslip 444, defines a flow channel 434 throughwhich the microfluidic volumes travel. The size and shape of the flowchannel 434 can be varied to suit a particular application. For example,the lower gasket 442 can be about 10 microns to about 3 millimeter (mm)thick, and can define an opening (flow channel 434) about 0.5 mm toabout 5 mm wide, and the length of the opening can nm substantially theentire width of the flow cell 430. In one embodiment, the lower gasket442 is about 50 microns thick and the flow channel 434 is about 1 mmwide by about 25 mm long. Alternatively, the microfluidic flow channel434 could be etched in the slide 440 and/or the coverslip 444.

In operation, the microfluidic volume is deposited into the flow cellinlet 448 on the connection ring 436 and is pulled through the flow cell430 under vacuum. The volume travels through the flow cell 430 as shownby the arrows in FIG. 10. Specifically, the volume travels downwardlythrough the connection ring 436 and through openings 439B, 443B in theupper gasket 438 and the slide 440, and then into the flow channel 434in the lower gasket 444. The volume then travels through the flowchannel 434 defined by the coverslip 444, the slide 440, and the lowergasket 442. Once the volume reaches the opposing opening 443A in theslide 440, the volume is drawn upwardly through the openings 443A, 439Ain the slide 440 and the upper gasket 438 and out the flow cell outlet446 by the vacuum pressure within, for example, the reservoir. Invarious embodiments, the slide 440 and/or coverslip 444 can be treatedto react with the microfluidic volume being pulled through the flow cell430. For example, a plurality of DNA strings can be adhered to thecoverslip in the area corresponding to the flow channel 434 in the lowergasket 442. Such an application is described in greater detail below.

One application for an apparatus in accordance with the inventionincludes performing single molecule sequencing. In this application, theflow cell includes individual strands of DNA or RNA (the template) boundto, for example, the coverslip 444 of the flow cell 430 (see FIGS. 9 and10). The DNA or RNA can be bound to the coverslip by any known means forbinding DNA or RNA to a surface using, for example, biotin-avidininteractions or other suitable attachment chemistries. A primer is addedthat hybridizes to a portion of the DNA or RNA bound in the flow cell.

The coverslip or other components of the flow cell that are exposed tothe flow path of the microfluidic volume can be produced and sold withspecific oligonucleotides bound thereto. Further, the coverslip materialcan include glass, quartz, silicon, or other materials present incommonly-available nucleic acid array chips. The material canincorporate an epoxide surface or another suitably reactive material tofacilitate binding of the DNA or RNA to the surface.

In one embodiment, the DNA or RNA to be sequenced is immobilized on theslide or coverslip using a biotin/streptavidin linkage. Alternatively,immobilization can occur via the primer. For example, a biotinylatedprimer can be immobilized on the coverslip via streptavidin linked tobiotin on the surface. Subsequent exposure of the immobilized primer tocomplementary DNA or RNA leads to sequence-specific hybridization withthe DNA or RNA strand to be sequenced.

Next, a microfluidic volume comprising a polymerase and a solution ofnucleotides is pulled through the flow cell and exposed to the boundtemplates. Complementary nucleotides will be incorporated in the primer.Detectable labels are used to improve detection. Detection, however, canoccur by detecting the indicia of nucleotide incorporation, for example,heat produced by the reaction or pyrophosphate production resulting fromincorporation. By monitoring nucleotide incorporation over time, theuser can thus determine the sequence of the exposed nucleotide at thatposition on the slide or coverslip. Because the apparatus permitsparallel monitoring of a very large number of individually-resolvablesingle molecules, each at a separate position on the coverslip, acorrespondingly large amount of sequence information can be collected atone time. Thus, computer systems are useful to monitor the observedlabel during the process and for handling the resulting sequence data.Depending on the nature of the DNA or RNA molecules sequenced, theapparatus can be used, for example, to identify nucleic acid sequencevariations associated with disease; to select or monitor a course oftreatment; or to monitor gene expression in an individual or in apopulation of individuals.

In another embodiment, single nucleotide detection is accomplished byattaching template nucleic acids to a flow cell in the presence of aprimer for template-dependent nucleic acid synthesis. Using a deviceaccording to the invention, a vacuum is created across the flow cell forintroduction of reagents for template-dependent nucleic acid synthesis.For example, once template/primer pairs are bound to the surface of theflow cell, reagents comprising labeled or unlabeled nucleotides and apolymerase to catalyze nucleotide addition are added via the flow cellinlet. The vacuum is switched on and the reagents are exposed to theflow cell and then exit via the flow cell outlet to the reservoir. Aftera wash step, complementary nucleotides added to primer are detected.Preferably, reagent nucleotides are labeled with, for example, afluorescent dye. Such dyes are observed using light microscopy. Forexample, cyanine dyes (cyanine-3 or cyanine-5) are useful for opticaldetection of incorporated nucleotides. Using optically-detectablelabels, nucleic acid sequencing is conducted on a single molecule level.This means that individual template nucleic acids are positioned on theflow cell such that each is individually optically resolvable. Thelocation of the templates is determined by, for example, the use ofdye-labeled primers that hybridize to individual templates. Labelednucleotides are flowed across the flow channel using the mechanismsdescribed herein under conditions that allow complementary nucleotideaddition to the primer. Once incorporated, the label is detected byexcitation of the dye at the appropriate wavelength and by using anemission filter for detection of the emission spectrum. Emissions thatoccur at a location known to contain a template indicate incorporationof the labeled base at that position. By conducting these steps multipletimes, a sequence is completed. Single molecule sequencing techniquesare described in Braslaysky, et al., PNAS (USA), 100: 3960-3964 (2003)and copending U.S. patent application Ser. No. 09/707,737.

A system for analyzing a sample in accordance with one embodiment of theinvention includes a lighting system 600. The lighting system 600, asshown in FIGS. 12A and 12B, may include a light source 602, a primaryfilter 604, a secondary filter 606, a shutter 608, a collimating lens609, a focusing lens 610, and a power source 612. A first portion of thelighting system 600, shown in FIG. 12A, includes three light sources602A, 602B, 602C (collectively 602). The lighting system 600, however,may include only two light sources or additional light sources asneeded. The light source 602 can include lasers, light emitting diodes,or lamps. In one embodiment, the first light source 602A has awavelength from about 390 nm to about 780 nm. In one embodiment, thefirst light source 602A is a red laser. The second light source 60213has a wavelength from about 936 nm to about 1340 nm. In one embodiment,the second light source 602B is an infrared laser. The third lightsource 602C has a wavelength from about 390 nm to about 780 nm. In oneembodiment, the third light source 602C is a green laser.

The lighting system 600 shown in FIG. 12A also includes three primaryfilters 604A, 604B, 604C (collectively 604). The primary filters 604 caninclude notch filters. The notch filters 604 are selected to transmitthe desired wavelength and to block unwanted wavelengths emitted by eachlight source 602. Additionally, the lighting system 600 shown in FIG.12A includes three secondary filters 606A, 606B, 606C (collectively606). The secondary filters 606 can include dichroic filters. In oneembodiment, the dichoric filters are placed at a 45° angle relative tothe light source 602. With a dichroic filter positioned at a 45° anglerelative to the light source 602, a light source that would have beentransmitted by the filter is still transmitted by the filter, but alight source that would have been blocked by the filter is reflected ata 90° angle. The lighting system 600 can also include shutter(s) 60 gfor blocking the light source(s) 602. Additionally, the focusing lens610 can be used for narrowing the beam emitted from the light source602, and the collimating lens 609 can be used for re-expanding andcollimating the beam from the light source 602 to the desired diameter.In one embodiment, the three light sources 602A, 602B, 602C arecollimated to substantially the same diameter. It is desirable for thebeams of the light sources 602 to be of substantially the same diameterand strength when they contact the sample of interest so that the fieldof illumination of the sample 620 is of equal size regardless of whichlight source 602 is used. Also, the lighting system 600 can include apower source 612 for providing power to the light sources 602. Thelighting system 600 can also include one or more mirrors for alteringthe optical path of the light sources as needed.

The lighting source 602 is directed to a desired point. As shown in FIG.12B, the first light source 602A can define a first optical path 630that intersects a sample of interest 620. The second light source 6028can be used to determine the position of the first optical path 630.Referring to FIG. 12A, the first light source 602A emits a beam of lightof a desired wavelength in a desired optical path 630. The beam of lightpasses through the focusing lens 610 that narrows the beam and thenthrough the collimating lens 609 that re-expands and collimates the beamto a desired diameter. The beam of the first light source 602A can beblocked by shutter 608 or allowed to pass through as desired. The beamof light passes through the notch filter 604A, where only the light ofthe desired wavelength is permitted to pass through. The beam of lightfrom the first light source 602A then reflects off the first dichroicfilter 606A at a 90° angle to the angle of incidence. The beam of lightfrom the first light source 602A passes through the subsequent ordownstream dichroic filters 606B, 606C in the desired optical path 630.

A second light source 602B emits a beam of light of a desiredwavelength. The beam of light then passes through notch filter 604B,where only the light of the desired wavelength is allowed to passthrough. The beam of light from the second light source 6028 thenreflects off the dichroic filter 606B at a 90° angle to the angle ofincidence, such that the beam of the second light source 6023 is atleast substantially coaxial (i.e., propogates along the same axis) withthe optical path 630 of the beam of the first light source 602A. Thebeams from the first light source 602A and the second light source 602Bhave substantially the same diameter. Both the beam from the first lightsource 602A and the beam from the second light source 606B pass throughthe third dichroic filter 606C.

A third light source 602C, which may be used in addition to or as analternative to the first light source 602A, emits a beam of light of adesired wavelength. The beam of light passes through the focusing lens610 that narrows the beam and then through the collimating lens 609 thatre-expands and collimates the beam to the desired diameter. The beam canbe blocked by the shutter 608 or allowed to pass through. The light thenpasses through the third notch filter 604C where only the light of thedesired wavelength is allowed to pass through. The beam of light fromthe third light source 602C then reflects off the third dichroic filter606C at a 90° angle to the angle of incidence, such that the beam of thethird light source 602C is at least substantially coaxial with the firstlight source 602A and/or the second light source 602B. The beam of thethird light source 602C has substantially the same diameter as the beamsfrom the first light source 602A and second light source 60213.

Because the first light source 602A and the third light source 602C canbe independently blocked, variations of which beams are directed to thedesired position are possible. For example, the third light source 602Ccan be blocked so that only the first light source 602A and the secondlight source 602B are directed to the desired point. Alternatively, allthree light sources 602A, 602B, 602C, can be directed to the desiredpoint at the same time. In some embodiments, the lighting system canalso include a neutral density filter 624 that is used to adjust thedensity of the light that is allowed to contact the sample 620. Forexample, if the sample 620 is saturated with light, the neutral densityfilter 624 can be adjusted to reduce the strength of the light directedto the sample 620. The neutral density filter 624 can be disposed alongthe optical path 630.

As shown in FIG. 12B, the optical path 630 of the coaxial beam of thelight source 602 is directed to a mirror 614 (or alternatively adichroic filter). The optical path is reflected at a 90° angle to theangle of incidence towards a filter 616. The beam from the first lightsource 602A and/or the third light source 602C reflects off filter 616at a 90° angle to the angle of incidence towards the sample of interest620. The beam of the second light source 60213 is refracted by thefilter 616 towards a position sensor 622 that senses the angle ofreflection of the optical path 630 relative to the sample 620.

The information provided by the position sensor 622 could be used toadjust the angle θ at which the optical path 630 of the light source602A intersects the sample 620. For example, the stage upon which thesample resides could be repositioned with respect to the optical path630 and/or the orientation of the mirror 614 could be adjusted.Alternatively, the lighting system 600 could include a translator 618that can be used to modify the angle of the optical path 630 of thelight source 602A towards the mirror 614. The translator 618 can includea micrometer that is used to set the desired angle θ of the optical path630.

The desired optical path 630 is one that results in total internalreflection of the beam of the light source 602A relative to the sampleof interest 620. The angle θ is the critical angle, and its valuedepends on the refractive indices of the media (θ=sin⁻¹ (densemedium/less-dense medium). Thus the angle θ depends on the density ofthe glass (i.e., “dense medium”), the quality of the surface of theglass, and the density of the sample (i.e., “less-dense medium”).

The position sensor 622 can be in communication with a computer, whichcan send a signal to automatically adjust the direction of the opticalpath 630 in response to a signal from the position sensor 622.Alternatively, the position sensor 622 could have a read out thatinforms the user of the angle of reflection θ of the optical path 630,which in turn could be manually adjusted. The angle θ of reflectance ofthe optical path 630 can be continuously monitored and adjusted asnecessary to maintain the critical angle θ, as the system operates.

When the light source 602 hits the sample 620 at the desired angle θ,all of the light is reflected (i.e., there is total internalreflection). Some of the energy of the beam, however, still propogates ashort distance into the less dense medium, generating an evanescentwave. A flourophore molecule attached to the sample of interest 620absorbs photons of the evanescent wave and is excited. The excitedfluorophores can be observed using, for example, an intensified CCDcamera.

The lighting system as illustrated in FIGS. 12A and 12B, and asdescribed above, is one possible arrangement of components of a lightingsystem in accordance with the invention. Other embodiments usingdifferent component arrangements, including different quantities andtypes of components such as filters and mirrors, are contemplated andconsidered within the scope of the invention. For example, multiplecomponents can be used for conditioning the light source and adjustingthe optical path or additional light sources could be used. Also,multiple sensors could be used to determine the angle of reflectance θof the optical path 630.

Having described certain embodiments of the invention, it will beapparent to those of ordinary skill in the art that other embodimentsincorporating the concepts disclosed herein may be used withoutdeparting from the spirit and scope of the invention. The describedembodiments are to be considered in all respects as only illustrativeand not restrictive.

1-8. (canceled)
 9. A lighting system for analyzing a sample, the systemcomprising: a first light source for analyzing a sample, light from thefirst light source defining a first optical path that intersects thesample; and a second light source operating with the first light sourcefor determining a position of the first optical path.
 10. The systemaccording to claim 9, wherein the sample is disposed within a flow cell.11. The system according to claim 9, wherein the first light source andthe second light source operate simultaneously.
 12. The system accordingto claim 9, wherein light from the second light source defines a secondoptical path at least partially coaxial with the first optical path. 13.The system according to claim 9, wherein the second light source isdirected to a position sensor for sensing an angle of incidence of thefirst optical path relative to the sample, and the relative orientationof the first optical path and the sample can be adjusted to vary theangle of incidence in response to a signal from the position sensor. 14.The system according to claim 9, wherein relative orientation of thefirst optical path and the sample can be adjusted to obtainsubstantially total internal reflection of the first light source at thesample.
 15. The system according to claim 9, wherein the first lightsource comprises a wavelength from about 390 nm to about 780 nm.
 16. Thesystem according to claim 9, wherein the second light source comprisesinfrared light.
 18. The system according to claim 9, wherein at leastone of the first light source and the second light source is selectedfrom the group consisting of a laser, a light emitting diode, and alamp.
 19. The system according to claim 9, wherein the system is usedfor single molecule detection.
 20. The system according to claim 9,wherein the system is coupled to an image capture device for capturingan image of the sample.
 21. A system for analyzing a sample, the systemcomprising: an optical instrument for viewing a sample; and a lightingsystem for illuminating the sample comprising one or more analyticallight sources, each light source defining an optical path thatintersects the sample, and a focusing light source operating with anyone of the analytical light sources to focus the optical instrument onthe sample.
 22. The system according to claim 21, wherein the sample isdisposed in a flow cell.
 23. The system according to claim 21, whereinthe optical system comprises two or more analytical light sources. 24.The system according to claim 21, wherein at least one of the analyticallight sources and the focusing light source operate simultaneously. 25.The system according to claim 21, wherein light from the focusing lightsource defines a second optical path at least partially coaxial with oneof the analytical optical paths.
 26. The system according to claim 21,wherein at least one of the analytical light sources emits light with awavelength from about 390 nm to about 780 nm.
 27. The system accordingto claim 21, wherein each of the analytical light sources emit light ofa different wavelength.
 28. The system according to claim 21, whereinthe focusing light source emits infrared light.
 29. The system accordingto claim 21, wherein the illumination and viewing employs total internalreflection fluorescence (TIRF).